Cooper is an essential element of human nutrition and is a critical component of proteins involved in cellular respiration, antioxidant defense and iron metabolism. The long-term objective of these studies is to define the molecular and cellular biology of intracellular copper transport and function. The specific aims of this proposal are intended to elucidate the mechanisms of copper delivery to Cu/Zn superoxide dismutase (SOD1). This enzyme is an essential component of antioxidant defense, functioning to catalyze the disproportionation of superoxide through the cyclic reduction of a bound copper ion. Gain-of-function mutations in human SOD1 have been linked to familial amyotrophic lateral sclerosis and evidence suggests that the neurodegeneration in this disease results from enhanced-free radical generating activity of the bound copper. Most recently we have identify the protein, termed CCS (chaperone for SOD1), which delivers copper to SOD1 and we now propose to define the molecular mechanisms by which this chaperone functions. Utilizing our knowledge of the structure of the murine CCS genomic locus, we now propose to generate a CCS null cell line to thus directly examine the role of CCS in copper delivery to SOD1. The expression of wild-type and mutant CCS in these cells, coupled with the availability of CCS specific antibodies, will permit clear definition of the CCS residues essential for cellular localization, SOD1 heterodimerization and copper transfer. These studies will provide new insights into the mechanisms of activation of SOD1 activity in cells and may provide novel therapeutic approaches to prevent or ameliorate neurodegeneration in patients with SOD1-linked amyotrophic lateral sclerosis.